We study the coordination of global patterns of bacterial gene expression by nutrient availability, continuing to focus on the roles of 3'-pyrophosphorylated analogs of GDP and GTP, called (p)ppGpp. Limiting growth for any of a variety of nutrients (amino acids, phosphate, nitrogen or energy sources) leads to changes (p)ppGpp levels that provoke regulatory adjustments at levels of transcription, translation, and metabolism. This year we have tried to learn more of the molecular details of how these nutritional signals regulate (p)ppGpp abundance. Regulation of (p)ppGpp synthesis by the RelA protein senses only a single nutrient: amino acids via the demands of protein synthesis for tRNA charged with amino acids. When charged tRNA is limiting, uncharged tRNA binds to codon-specified ribosomal acceptor (A) sites and somehow activates synthesis of (p)ppGpp by RelA residing on the ribosome. Last year, we mapped the dimethylsulfate footprint of RelA bound to ribosomes to regions near the CCA-3'OH end of tRNA and the A site codon-anticodon. This year we found that these RelA binding sites are near several RNA-RNA bridges and probably weaken interactions between small and large subunits to an extent that is inconsistent with peptide bond formation. Furthermore, salt requirements for optimal RelA binding and ppGpp synthesis are also incompatible with peptidyl transferase activity. This implies something new; the binding of RelA and its activation for the synthesis of (p)ppGpp on a ribosome idling for lack of appropriate charged tRNA may be mechanistically linked with a failure to form a peptide bond. We are developing a system for immobilizing functional ribosomes on a solid support to facilitate analysis of binding of tRNA and RelA during synchronized steps in protein synthesis. Regulation of (p)ppGpp synthesis by the SpoT protein senses nutrients other than amino acids by unknown mechanisms. We have initiated a promising bacterial two hybrid screen for proteins that interact with the SpoT C-terminal region containing TGS and ACT domains. In addition, we have constructed a SpoT mutant by deleting two key residues in the pyrophosphohydrolase catalytic center yielding an enzyme devoid of (p)ppGppase activity that retains (p)ppGpp synthetic activity. This mutant provides a probe for disentangling regulatory effects on SpoT, a bifunctional enzyme.